Protein purification

ABSTRACT

The present invention relates to a process for the purification of an antibody fragment from a periplasmic cell extract comprising a first cation exchange chromatography step and a second anion exchange chromatography step.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of InternationalPatent Application No. PCT/EP2011/062837, filed Jul. 26, 2011, thedisclosure of which is hereby incorporated by reference in its entirety,including all figures, tables and amino acid or nucleic acid sequences.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Jan. 4, 2013 and is 8 KB. The entire contents ofthe sequence listing is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention is in the field of protein purification. Morespecifically, it relates to a process for the purification of antibodyfragments.

BACKGROUND OF THE INVENTION

The large-scale, economic purification of proteins is increasingly animportant problem for the biotechnology industry. Generally, proteinsare produced by cell culture, using either mammalian or bacterial celllines engineered to produce the protein of interest by insertion of arecombinant plasmid containing the gene for that protein. Since the celllines used are living organisms, they must be fed with a complex growthmedium, containing sugars, amino acids, and growth factors. The proteinof interest must be isolated from the mixture of compounds fed to thecells and from the by-products of the cells themselves (feed stream) topurity sufficient for use as a human therapeutic. The standards set byhealth authorities for proteins intended for human administrationregarding impurities from the feed stream are very high. Manypurification methods for proteins known in the art contain stepsrequiring the application e.g. of low or high pH, high saltconcentration or other extreme conditions that may irreversiblyjeopardize the biological activity of the protein to be purified and aretherefore not suitable. Thus, separation of the desired protein tosufficient purity poses a formidable challenge. Historically, proteinpurification schemes have been predicated on differences in themolecular properties of size, charge and solubility between the proteinto be purified and undesired protein contaminants. Protocols based onthese parameters include size exclusion chromatography, ion exchangechromatography, differential precipitation and the like.

Antibodies and antibody fragments are of increasing importance in arange of therapeutic areas. One of the most important methods ofproducing antibodies antibody fragments is by recombinant technology.Such techniques use a host cell to express the desired antibody, orantibody fragment, which is then separated from the production mediumand purified.

Antibodies require glycosylation and are therefore generally expressedin eukaryotic expression systems employing eukaryotic cells, inparticular mammalian cells such as CHO, PER.C6, NS0, BHK or Sp2/0 cells.In eukaryotic expression systems the protein of interest expressed suchas an antibody is generally secreted into the cell culture medium. Themedium can subsequently be separated easily from the protein secretingcells, e.g. by centrifugation or filtration.

Almost all current industrial antibody purification platforms useprotein A (described e.g. in WO 98/23645). Protein A is a cell surfaceprotein found in the cell wall of the bacteria staphylococcus aureusthat binds to the Fc portion of mammalian immunoglobulin. Protein A hasa high affinity to human IgG₁ and IgG₂ and a moderate affinity to humanIgM, IgA and IgE antibodies. Consequently, protein A purification is notwell suited for antibody fragments that lack the Fc portion.

A protein that does not require glycosylation is preferably expressed inprokaryotic expression systems employing prokaryotic cells such asgram-negative bacteria. Particularly, an antibody that does not requireglycosylation, for example an antibody fragment such as a Fab, an Fab′or a scFv is preferably expressed in such systems. Prokaryoticexpression systems and in particular Escherichia coli (E. coli) systemsor other gram-negative bacteria allow the manufacturing of proteins thatdo not require glycosylation, such as for example antibody fragments, inan economically attractive way. Manufacturing of proteins in E. coli isbeneficial in particular due to due to lower costs of goods and fasterdrug development processes (Humphreys, 2003; Humphreys, 2003).Prokaryotic and in particular E. coli protein expression systems arewell known in the art (Swartz, 2001; Jana and Deb, 2005; Terpe, 2006).Prokaryotic cells do not actively secrete a heterologous protein ofinterest expressed in the cell. Gram-negative prokaryotic cells such asE. coli, however, can be engineered such that heterologous proteinsexpressed in the cell, such as antibody fragments, are exported into theperiplasmic space where they can form disulfide bonds. Isolation ofthese heterologous proteins from the periplasmic space requires thedisruption of the outer membrane of the prokaryotic cells which resultsin substantial release also of host cell proteins (HCPs). Methods fordisrupting the outer membrane of a gram-negative prokaryotic cell andsubsequent harvest of the cell culture fluid containing the heterologousare well known in the art. Manufacturing of antibody fragments in E.coli also results in the production of by-products such as truncatedlight chains, gluthathione adducts of light chains and light chaindimers (Battersby et al., 2001).

Cell culture fluid (feed stream) harvested from prokaryotic expressionsystems such as E. coli expression systems and in particular periplasmiccell extract from gram-negative bacteria differs substantially from cellculture fluid harvested from eukaryotic expression systems in therelative amount and composition of HCPs, bacterial DNA and endotoxinwhich need to be separated from the heterologous protein of interestthat is expressed in the prokaryotic or eukaryotic expression system.Concentration of HCP as well as complexity and heterogeneity of HCPdepend on the expression system or cell line and the cell cultureconditions (Arunakumari, 2007). Purification of antibody fragmentsexpressed in prokaryotic expression systems, in particular ingram-negative prokaryotic expression systems, faces therefore adifferent set of challenges and requires different approaches (Humphreysand Glover, 2001). Basic principles of purification of monoclonalantibody fragments are known in the art (Spitali, 2009). There are twomedicinal products currently approved by the US Food and DrugAdministration (FDA) and the European Medicines Agency (EMA) whichcomprise an antibody fragment as active ingredient which is produced inmicrobial cells: certolizumab pegol (Cimzia®) comprises a Fab bindingspecifically to TNFα and ranibizumab (Lucentis®) is a Fab fragmentbinding specifically to vascular endothelial growth factor (VEGF).Purification of ranibizumab from microbial feed stream is performedusing a process with four chromatography steps (Walsh, 2007). Themedicinal product abciximab (ReoPro®) comprises the Fab fragment of thechimeric human-murine monoclonal antibody 7E3 which binds to theglycoprotein (GP) IIb/IIIa receptor of human platelets and inhibitsplatelet aggregation. The chimeric 7E3 antibody is produced bycontinuous perfusion in mammalian cell culture. The 48 Kd Fab fragmentis obtained from the purified full length antibody after digestion withpapain and column chromatography.

Affinity chromatography separates proteins on the basis of a reversibleinteraction between a protein (or group of proteins) of interest and aspecific ligand coupled to a chromatography matrix. The interactionbetween the protein of interest and ligand coupled to the chromatographymatrix can be a result of electrostatic or hydrophobic interactions, vander Weals' forces and/or hydrogen bonding. To elute the target moleculefrom the affinity medium the interaction can be reversed, eitherspecifically using a competitive ligand, or non-specifically, bychanging the pH, ionic strength or polarity. Affinity purificationrequires a biospecific ligand that can be covalently attached to achromatography matrix. The coupled ligand must retain its specificbinding affinity for the target molecules and, after washing awayunbound material, the binding between the ligand and target moleculemust be reversible to allow the target molecules to be removed in anactive form. Despite its common use, affinity chromatography is costly,particularly at the industrial scale necessary to purify therapeuticproteins.

Ion exchange chromatography can be used to purify ionizable molecules.Ionized molecules are separated on the basis of the non-specificelectrostatic interaction of their charged groups with oppositelycharged molecules attached to the solid phase support matrix, therebyretarding those ionized molecules that interact more strongly with solidphase. The net charge of each type of ionized molecule, and its affinityfor the matrix, varies according to the number of charged groups, thecharge of each group, and the nature of the molecules competing forinteraction with the charged solid phase matrix. These differencesresult in resolution of various molecule types by ion-exchangechromatography. Elution of molecules that are bound to the solid phaseis generally achieved by increasing the ionic strength (i.e.conductivity) of the buffer to compete with the solute for the chargedsites of the ion exchange matrix. Changing the pH and thereby alteringthe charge of the solute is another way to achieve elution of thesolute. The change in conductivity or pH may be gradual (gradientelution) or stepwise (step elution). Two general types of interactionare known: Anionic exchange chromatography mediated by negativelycharged amino acid side chains (e.g. aspartic acid and glutamic acid)interacting with positively charged surfaces and cationic exchangechromatography mediated by positively charged amino acid residues (e.g.lysine and arginine) interacting with negatively charged surfaces. Anionexchangers can be classified as either weak or strong. The charge groupon a weak anion exchanger is a weak base, which becomes de-protonatedand, therefore, loses its charge at high pH. Diethylaminoethyl(DEAE)-cellulose is an example of a weak anion exchanger, where theamino group can be positively charged below pH˜9 and gradually loses itscharge at higher pH values. DEAE or diethyl-(2-hydroxy-propyl)aminoethyl(QAE) have chloride as counter ion, for instance.

An alternative to elution by increase in ion strength of the elutionbuffer (elution chromatography) is elution using of molecules which havea higher dynamic affinity for the stationary phase than the boundprotein. This mode of performing ion-exchange chromatography is calleddisplacement chromatography. Displacement chromatography isfundamentally different from any other modes of chromatography in thatthe solutes are not desorbed in the mobile phase modifier and separatedby differences in migration rates (Tugcu, 2008). In displacement,molecules are forced to migrate down the chromatographic column by anadvancing shock wave of a displacer molecule that has a higher affinityfor the stationary phase than any component from the feed stream. It isthis forced migration that results in higher product concentrations andpurities compared to other modes of operation of high retention,followed by a constant infusion of a displacer solution into the column.

Dynamic binding capacity describes the amount of protein of interestwhich will bind to a chromatography resin in a column under defined flowconditions. The dynamic binding capacity for a chromatography resin isdependent on running conditions (e.g. flow rate, pH and conductivity),origin of the sample, sample preparation and the other bindingimpurities present. Dynamic binding capacities are determined by loadinga sample containing a known concentration of the protein of interest,and monitoring the concentration in the column flow-through (Do et al.,2008). The dynamic binding capacity of an ion exchange resin is definedas the point during loading when the protein of interest starts to berecovered in the flow-through. Typically a value of 10% for theproportion of protein of interest in the flow-through compared to theload is used to define this point (McCue et al., 2003). For impurityremoval, the threshold for the impurity in the flow-through is setaccording to criteria specific to the application.

WO 99/57134, WO 2004/024866 and WO 2007/117490 relate to processes forprotein or antibody purification comprising ion exchange chromatography.The processes are exemplified using antibodies produced in mammaliancells. WO 2009/058812 relates to a process for antibody purificationcomprising cation exchange chromatography. The process is exemplifiedusing antibodies produced in mammalian cells. WO 2007/108955 relates toa two-step non-affinity ion exchange chromatograph process for proteinpurification comprising cation exchange chromatography followed by ionexchange chromatography. The Example in WO 2007/108955 describes thepurification of fully human antibody produced in mammalian cells.Multiple washing steps were performed during cation exchangechromatography and the eluate diluted prior to anion exchangechromatography. Humphreys et al. describes the purification of Fab′ at alaboratory scale using cation exchange chromatography and ion exchangechromatography (Humphreys et al., 2004). WO 2004/035792 relates to thegeneration of E. coli strains expressing mutant PhoS protein in order toreduce PhoS protein impurities in antibody fragment preparationspurified from bacterial cell culture.

CDP870 is a genetically engineered antibody fragment (Fab′) chemicallylinked to a PEG moiety as described in WO 01/94585 (which isincorporated herein by reference in its entirety). CDP870 has potenthuman TNFα neutralizing properties.

There is a need in the art for methods of purifying antibody fragmentsfrom cell culture fluids harvested from prokaryotic and in particulargram-negative bacteria such as E. coli expression systems. There isparticular need in the art for methods of purifying antibody fragmentsfrom periplasmic cell extracts harvested from prokaryotic and inparticular gram-negative bacteria such as E. coli expression systemsthat are suitable to operate with cell extracts that contain a very hightiter of antibody fragment or HCP or both antibody fragment and HCP.High titer expression of the antibody fragments require methods suitablefor the purification of the large quantities of antibody fragments in aneconomical manner: reducing the column sizes, buffer usage andprocessing times (GE Healthcare data file 11-0025-76 AE, 2007).

SUMMARY OF THE INVENTION

Purification requirements and challenges differ substantially forproteins that have purified from bacterial cell culture from proteinsthat have to be purified from eukaryotic cell culture. Particulardifficulties are faced when proteins need to be purified fromperiplasmic cell extracts of gram-negative bacteria due to e.g. theamount of bacterial host cell proteins present. Further difficultieshave to be overcome when proteins needs be purified from gram-negativebacterial cultures that express heterologous proteins to a very highconcentration.

The inventors have surprisingly found a new process for purification ofan antibody fragment from a periplasmic cell extract wherein the processis highly efficient and suitable for periplasmic cell extract comprisingantibody fragment at a high concentration.

In aspect the invention provides a process for the purification of anantibody fragment from a periplasmic cell extract comprising a firstchromatography step to capture the antibody fragment wherein a mixturecontaining an antibody fragment is subjected to cation exchangechromatography and subsequently eluted to produce a first eluatecontaining the antibody fragment; and a second chromatography stepwherein the first eluate is subjected to anion exchange chromatographyto capture impurities and produce a flow through containing the antibodyfragment, and recovering said antibody fragment.

In another aspect the invention provides a process for the purificationof an antibody fragment from a periplasmic cell extract consistingessentially of a first chromatography step to capture the antibodyfragment wherein a mixture containing an antibody fragment is subjectedto cation exchange chromatography to subsequently eluted to produce afirst eluate containing the antibody fragment; a first ultrafiltrationis applied to the first eluate; a second chromatography step wherein thepurified first eluate is subjected to anion exchange chromatography tocapture impurities to produce a flow through containing the antibodyfragment; and a second ultrafiltration applied to the flow through, andrecovering said antibody fragment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a chromatogram of protein (including Fab′) monitored byabsorbance of UV light [measured in milli-absorbance units (mAU)] (solidline) along the conductivity (dotted line) from the first capture stepon a cation exchange chromatography column (Capto S™). The chromatogramshows that a large volume can be loaded onto column during which someproteins do not bind, followed by the recovery of bound proteinsincluding the Fab′ in a small volume with an increase in conductivity.

FIG. 2 shows a chromatogram from the step on an anion exchangechromatography column (Capto Q™). The chromatogram shows the non-bindingof a Fab′ and its appearance in the post-load wash with the boundimpurities recovered in the regeneration peak.

FIG. 3 shows an SDS-PAGE analysis of capture load, capture eluate andanion exchange flow-through.

FIG. 4 shows Western-Blot to detect host cell proteins present in thesample before (Lane 2) and after (Lane 3) the anion exchangechromatography.

FIG. 5 shows the amino acid sequences of CDP870.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the amino acid sequence of CDRH1 of CDP870.

SEQ ID NO: 2 shows the amino acid sequence of CDRH2 of CDP870.

SEQ ID NO: 3 shows the amino acid sequence of CDRH3 of CDP870.

SEQ ID NO: 4 shows the amino acid sequence of CDRL1 of CDP870.

SEQ ID NO: 5 shows the amino acid sequence of CDRL2 of CDP870.

SEQ ID NO: 6 shows the amino acid sequence of CDRL3 of CDP870.

SEQ ID NO: 7 shows the nucleotide and predicted amino acid sequence ofthe light chain variable region CDP870.

SEQ ID NO: 8 shows the nucleotide and predicted amino acid sequence ofthe heavy chain variable region CDP870.

SEQ ID NO: 9 shows the amino acid sequence of a grafted anti-TNFα FabCDP870 light chain.

SEQ ID NO: 10 shows the amino acid sequence of a grafted anti-TNFα FabCDP870 heavy chain.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the invention relates to a process for thepurification of an antibody fragment from a periplasmic cell extractcomprising a first chromatography step to capture the antibody fragmentwherein a mixture, such as a periplasmic cell extract, containing anantibody fragment is subjected to cation exchange chromatography andsubsequently eluted to produce a first eluate containing the antibodyfragment; and a second chromatography step wherein the first eluate issubjected to anion exchange chromatography to capture impurities andproduce a flow through containing the antibody fragment, and recoveringsaid antibody fragment.

In a second aspect the invention relates to a process for thepurification of an antibody fragment from a periplasmic cell extractconsisting essentially of a first chromatography step to capture theantibody fragment wherein a mixture, such as a periplasmic cell extractcontaining an antibody fragment is subjected to cation exchangechromatography to subsequently eluted to produce a first eluatecontaining the antibody fragment; a first ultrafiltration applied to thefirst eluate; a second chromatography step wherein the purified firsteluate is subjected to anion exchange chromatography to captureimpurities to produce a flow through containing the antibody fragment;and a second ultrafiltration applied to the flow through, and recoveringsaid antibody fragment.

In a first embodiment of the first aspect of the invention the processaccording to the first aspect of the invention comprises only twochromatography steps.

In a first embodiment of the second aspect of the invention the processaccording to the second aspect of the invention the ultrafiltrationafter the cation exchange chromatography and the ultrafiltration afterthe anion exchange chromatography are performed by tangential flowfiltration (TFF).

In a second embodiment of the first or second aspect of the invention inthe process according to the first embodiment of the first or secondaspect of the invention all chromatography steps are performed on achromatography column.

In a third embodiment of the first or second aspect of the invention inthe process according to the first or second embodiment of the first orsecond aspect of the invention the cation exchange chromatography of thefirst chromatography step is performed in elution mode.

In a fourth embodiment of the first or second aspect of the invention inthe process according to the second or third embodiment of the first orsecond aspect of the invention the cation exchange chromatography of thefirst chromatography step comprises the following steps in sequentialorder:

-   -   a) loading a mixture, such as a periplasmic cell extract,        containing an antibody fragment onto the cation exchange column,    -   b) washing the cation exchange column with a wash buffer wherein        during the washing the conductivity, pH and salt concentration        of the wash buffer is not changed, and    -   c) eluting the antibody fragment with an elution buffer.

In a fifth embodiment of the first or second aspect of the invention inthe process according to the fourth embodiment of the first or secondaspect of the invention the pH of the washing buffer is identical to thepH of the mixture, such as a periplasmic cell extract, containing anantibody fragment, prior to the first chromatography step.

In a sixth embodiment of the first or second aspect of the invention inthe process according to the first, second, third, fourth or fifthembodiment of the first or second aspect of the invention the mixture,such as a periplasmic cell extract, containing an antibody fragment,prior to the first chromatography step has a pH of between 4.0 to 5.0,preferably a pH of between 4.3 to 4.7, more preferably a pH of between4.3 to 4.5 and most preferably 4.5.

In a seventh embodiment of the first or second aspect of the inventionin the process according to the first, second, third, fourth, fifth orsixth embodiment of the first or second aspect of the invention themixture containing an antibody fragment and being subjected to cationexchange chromatography as a primary capture step contains total proteinat a concentration of at least 1.5 g/L, or at least 3 g/L, or at least 4g/L, or at least 5 g/L, or at least 7.5 g/L, or at least 10 g/L or atleast 20 g/L, or at least 40 g/L, or at a concentration of between 3 and40 g/L, or at a concentration of between 4 and 20 g/L, or at aconcentration of between 5 and 15 g/L.

In an eighth embodiment of the first or second aspect of the inventionin the process according to the first, second, third, fourth, fifth,sixth or seventh embodiment of the first or second aspect of theinvention the mixture containing an antibody fragment and beingsubjected to cation exchange chromatography as a primary capture stepcontains antibody fragment at a concentration of at least 3 g/L, or atleast 4 g/L, or at least 5 g/L, or at least 7.5 g/L, or at least 10 g/Lor at least 20 g/L, or at a concentration of between 3 and 20 g/L, or ata concentration of between 4 and 50 g/L, or at a concentration ofbetween 5 and 10 g/L.

In a ninth embodiment of the first or second aspect of the invention inthe process according to the first, second, third, fourth, fifth, sixth,seventh or eighth embodiment of the first or second aspect of theinvention the cation exchange chromatography of the primary capture stepis performed at a flow rate of at least 300 cm/h, preferably between 300and 2000 cm/h, more preferably, between 350 and 1500 cm/h, even morepreferably between 350 and 1000 cm/h, and most preferably between 400and 700 cm/h.

In a tenth embodiment of the first or second aspect of the invention inthe process according to the first, second, third, fourth, fifth, sixth,seventh, eighth or ninth embodiment of the first or second aspect of theinvention the cation exchange chromatography of the primary capture stepis performed at a conductivity of not more than 6 mS/cm, preferablybetween 6 and 2 mS/cm, more preferably between 5 and 3 mS/cm, and evenmore preferably between 4.5 and 3.5 mS/cm.

In an eleventh embodiment of the first or second aspect of the inventionin the process according to the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth or tenth embodiment of the first or secondaspect of the invention the cation exchange chromatography of theprimary capture step is performed on a resin comprising sulphonyl,sulphopropyl or carboxymethyl coupled to a resin of suitable materialknown in the art, including but not limited to crosslinked, beaded-formsof agarose (e.g. Sepharose™ or Superose™), modified methacrylatepolymers (e.g. tentacle, hydroxylated); silica; ceramic and styrenedivinylbenzene.

In a twelfth embodiment of the first or second aspect of the inventionin the process according to the eleventh embodiment of the first orsecond aspect of the invention the resin of the cation exchangechromatography of the primary capture has a dynamic binding capacity forthe antibody fragment of at least 50 g/L of resin, or at least 60 g/L ofresin, or at least 75 g/L of resin, or at least 150 g/L resin, orbetween 50 and 150 g/L resin, or between 60 and 100 g/L resin, orbetween 50 and 75 g/L resin.

In an thirteenth embodiment of the first or second aspect of theinvention in the process according to the eleventh or the twelfthembodiment of the first or second aspect of the invention the resin ofthe cation exchange chromatography of the primary capture has a meanparticular size of at least 50 μm, preferably between 60 and 300 μm,more preferably between 70 and 200 μm, and even more preferably between80 and 100 μm.

In a fourteenth embodiment of the first or second aspect of theinvention in the process according to the first, second, third, fourth,fifth sixth, seventh, eighth, ninth, tenth, eleventh, twelfth orthirteenth embodiment of the first or second aspect of the invention themixture containing an antibody fragment which is subjected to cationexchange chromatography in the primary capture step contains bacterialhost cell protein in an amount of between about 200 μg/ml to 10,000μg/ml, about 500 μg/ml to 5000 μg/ml, about 1000 μg/ml to 4000 μg/ml orabout 2000 μg/ml to 4000 μg/ml.

In a fifteenth embodiment of the first or second aspect of the inventionin the process according to the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth orfourteenth embodiment of the first or second aspect of the invention thein the cation exchange chromatography of the primary capture stepbetween 5 and 100 g antibody fragment per liter resin, between 10 and 90g antibody fragment per liter resin or between 20 and 75 g antibodyfragment per liter resin are loaded.

In a sixteenth embodiment of the first or second aspect of the inventionin the process according to the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth,fourteenth or fifteenth embodiment of the first or second aspect of theinvention the anion exchange chromatography in the second chromatographystep is performed on a resin comprising quaternary ammonium (Q),diethylaminoethyl (DEAE) or trimethylaminoethyl (TMAE) coupled to aresin of suitable material known in the art, including but not limitedto crosslinked, beaded-forms of agarose (e.g. Sepharose™ or Superose™),modified methacrylate polymers (e.g. tentacle, hydroxylated); silica;ceramic and styrene divinylbenzene. The second chromatography step mayalso be performed on a membrane comprising a quaternary ammonium orpoly(allylamine) coupled to a membrane of suitable material known to theart, including but not limited to cellulose and polyethylene.

In a seventeenth embodiment of the first or second aspect of theinvention in the process according to the first, second, third, fourth,fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,thirteenth, fourteenth, fifteenth or sixteenth embodiment of the firstor second aspect of the invention the anion exchange chromatographycolumn in the second chromatography step has a mean particular size ofat least 50 μm, preferably between 60 and 300 μm, and more preferablybetween 70 and 200 μm, and even more preferably between 80 and 100 μm.

In a eighteenth embodiment of the first or second aspect of theinvention in the process according to the first, second, third, fourth,fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,thirteenth, fourteenth, fifteenth, sixteenth or seventeenth embodimentof the first or second aspect of the invention the anion exchangechromatography in the second chromatography step is performed at a pH ofbetween 6 to 10, preferably a pH of between 7 to 9, more preferablybetween 8 to 9, and even more preferably at a pH of 8.5.

In an nineteenth embodiment of the first or second aspect of theinvention in the process according to the first, second, third, fourth,fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,thirteenth, fourteenth, fifteenth, sixteenth, seventeenth or eighteenthembodiment of the first or second aspect of the invention the anionexchange chromatography in the second chromatography step is performedhas a binding capacity for the process-related impurities of greaterthan 20 g/L of resin, preferably greater than 30 g/L of resin, and evenmore preferably greater than 40 g/L of resin, or between 20 and 80 g/Lresin, or between 20 and 40 g/L resin.

In a twentieth embodiment of the first or second aspect of the inventionthe process according to the first, second, third, fourth, fifth, sixth,seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth,fourteenth, fifteenth, sixteenth, seventeenth, eighteenth or nineteenthembodiment of the first or second aspect of the invention does notcomprise a high performance tangential flow filtration (HPTFF) step.

In a twenty-first embodiment of the first or second aspect of theinvention in the process according to the first, second, third, fourth,fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth,nineteenth or twentieth embodiment of the first or second aspect of theinvention the antibody fragment recovered contains host cell protein(HCP) in an amount of not more than 150 parts per million (ppm), or notmore than 120 ppm or not more than 100 ppm.

In further embodiments the process according to any of the embodimentsof the first or second aspect of the invention wherein the antibodyfragment is a Fab, Fab′, F(ab′)₂, Fv, a scFv or camelid antibody.

The term “affinity chromatography” as used herein, refers to a proteinseparation technique in which a protein of interest or antibody ofinterest is reversibly and specifically bound to a biospecific ligand.Preferably, the biospecific ligand is covalently attached to achromatographic solid phase material and is accessible to the protein ofinterest in solution as the solution contacts the chromatographic solidphase material. The protein of interest (e.g. an antibody) retains itsspecific binding affinity for the biospecific ligand (antigen,substrate, cofactor, or hormone, for example) during the chromatographicsteps, while other solutes and/or proteins in the mixture do not bindappreciably or specifically to the ligand. Binding of the protein ofinterest to the immobilized ligand allows contaminating proteins orprotein impurities to be passed through the chromatographic medium whilethe protein of interest remains specifically bound to the immobilizedligand on the solid phase material. The specifically bound protein ofinterest is then removed in active form from the immobilized ligand withlow pH, high pH, high salt, competing ligand, and the like, and passedthrough the chromatographic column with the elution buffer, free of thecontaminating proteins or protein impurities that were earlier allowedto pass through the column. Any substance can be used as a ligand forpurifying its respective specific binding protein, e.g. antibody.

The terms “aglycosylated” and “non-glycosylated” are usedinterchangeably herein and refer to the lack of specificpost-translational addition of a glycosyl- or carbohydrate moiety to aprotein such as an antibody.

The term “antibody” or “antibodies” as used herein, refers to monoclonalor polyclonal tetrameric full length antibodies comprising two heavy andtwo lights chains.

The term immunoglobulin or immunoglobulins is used synonymously with“antibody” or “antibodies”, respectively. The term “antibody” or“antibodies” as used herein includes but is not limited to recombinantantibodies that are generated by recombinant technologies as known inthe art. An “antibody” or “antibodies” can be of any origin includingfrom mammalian species such as human, non-human primate (e.g. human suchas from chimpanzee, baboon, rhesus or cynomolgus monkey), rodent (e.g.from mouse, rat, rabbit or guinea pig), goat, bovine or horse species.The antibody herein is directed against an “antigen” of interest.Preferably, the antigen is a biologically important polypeptide andadministration of the antibody to a mammal suffering from a disease ordisorder can result in a therapeutic benefit in that mammal. However,antibodies directed against non-polypeptide antigens are alsocontemplated. Where the antigen is a polypeptide, it may be atransmembrane molecule (e.g. receptor) or ligand such as a growth factoror cytokine. Preferred molecular targets for antibodies encompassed bythe present invention include CD polypeptides such as CD3, CD4, CD8,CD19, CD20, CD22, CD34, CD38, CD40 and CD40-L; FcRN; OX40; members ofthe HER receptor family such as the EGF receptor, HER2, HER3 or HER4receptor; cell adhesion molecules such as LFA-1, Mac1, p150,95, VLA-4,ICAM-1, VCAM and av/b3 integrin including either α or β subunits thereof(e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies); chemokines andcytokines or their receptors such as IL-1α and β, IL-2, IL-6, the IL-6receptor, IL-12, IL-13, IL-17 forms, IL-18, IL-21, IL-23, TNFα and TNFβ;growth factors such as VEGF; IgE; blood group antigens; flk2/flt3receptor; obesity (OB) receptor; mpl receptor; CTLA-4; polypeptide C;etc.

The term “antibody fragment” or “antibody fragments” as used herein,refers an aglycosylated antibody or an aglycosylated portion of anantibody, generally the antigen binding or variable region thereof.Examples of antibody fragments include any antibody that lacks the orhas no Fc portion. Examples of antibody fragments include also Fab,Fab′, F(ab′)₂, Fv and scFv fragments; diabodies; triabodies;tetrabodies; minibodies; antibodies consisting essentially of a single,two or three immunoglobulin domain(s) such as Domain Antibodies™;single-chain antibodies; bispecific, trispecific, tetraspecific ormultispecific variants of any of the above. The term “antibody fragment”or “antibody fragments” as used herein also refers to camelid antibodies(e.g. from camels or llamas such as Nanobodies™) and derivativesthereof. Antibodies fragments are well known in the art (Holliger andHudson, 2005). Various techniques have been developed for the productionof antibody fragments and are known in the art (Glover and Humphreys,2004). The term “antibody fragment” or “antibody fragments” as usedherein, comprises human, humanized, primatized and chimeric antibodyfragments.

The term “buffer” as used herein, refers to a substance which, by itspresence in solution, increases the amount of acid or alkali that mustbe added to cause unit change in pH. A buffered solution resists changesin pH by the action of its acid-base conjugate components. Bufferedsolutions for use with biological reagents are generally capable ofmaintaining a constant concentration of hydrogen ions such that the pHof the solution is within a physiological range. Traditional buffercomponents include, but are not limited to, organic and inorganic salts,acids and bases.

The term “chromatography” as used herein, refers to the process by whicha substance of interest in a mixture is separated from other substancesin a mixture as a result of differences in rates at which the individualsolutes of the mixture migrate through a stationary medium under theinfluence of a moving phase, or in bind and elute processes.

The term “chromatography column” or “column” in connection withchromatography as used herein, refers to a container, frequently in theform of a cylinder or a hollow pillar which is filled with thechromatography matrix or resin. The chromatography matrix or resin isthe material which provides the physical and/or chemical properties thatare employed for purification.

The term “conductivity” as used herein, refers to the ability of anaqueous solution to conduct an electric current between two electrodes.In solution, the current flows by ion transport. Therefore, with anincreasing amount of ions present in the aqueous solution, the solutionwill have a higher conductivity. The unit of measurement forconductivity is milliSiemens per centimeter (mS/cm), and can be measuredusing a conductivity meter.

The conductivity of a solution may be altered by changing theconcentration of ions therein. For example, the concentration of abuffering agent and/or concentration of a salt (e.g. NaCl or KCl) in thesolution may be altered in order to achieve the desired conductivity.

The term “eluate” as used herein, refers to a liquid compositioncomprising the substance, (e.g. the antibody fragment or contaminantsubstance) which was obtained subsequent to the binding of saidsubstance to a chromatography material and addition of an elution bufferto dissociate the substance from the chromatography material. Eluatesmay be referred to with respect to the step in the purification process.For example, the term “first eluate” refers to the eluate from the firstchromatographic step; the term “second eluate” refers to the eluate fromthe second chromatographic step, etc.

The term “flow-through” as used herein, refers to a liquid compositioncomprising the substance, (e.g. the antibody fragment or contaminantsubstance) which was obtained by passing a mixture comprising saidsubstance over a chromatography material such that the molecule passesover the material without binding.

The term “mixture”, as used herein, refers to an at least partiallyliquid composition comprising at least one antibody fragment of interestwhich is sought to be purified from other substances which may also bepresent. Mixtures can, for example, be suspensions, aqueous solutions,organic solvent systems, or aqueous/organic solvent mixtures orsolutions. The mixtures are often complex mixtures or solutionscomprising many biological molecules (such as proteins, antibodies,hormones, and viruses), small molecules (such as salts, sugars, lipids,etc.) and even particulate matter. While a typical mixture of biologicalorigin may begin as an aqueous solution or suspension, it may alsocontain organic solvents used in earlier separation steps such assolvent precipitations, extractions, and the like.

The term “periplasmic cell extract” as used herein, refers to thecomposition obtained from a cell culture of gram negative prokaryoticcells following the disruption of the outer membrane and release ofmaterial from the periplasmic space. A periplasmic cell extract isfrequently liquid and may also contain particular matter or gas. Theterm “periplasmic cell extract” also includes a liquid composition thatmay have been treated further following the collection from theperiplasmic space, e.g. to remove insoluble material.

The term “purification” or “purifying” or “purified” refers to a processwherein from a mixture containing a protein of interest, such as anantibody or antibody fragment, unwanted substances such as HCPs, DNA orsalts are removed or reduced. A “purification step” may be part of anoverall purification process resulting in a “homogeneous” composition,which is used herein to refer to a composition comprising less than 150ppm HCP in a composition comprising the protein of interest,alternatively less than 120 ppm, less than 100 ppm, less than 90 ppm,less than 80 ppm, less than 70 ppm, less than 60 ppm, less than 50 ppm,less than 40 ppm, less than 30 ppm, less than 20 ppm, less than 10 ppm,less than 5 ppm, or less than 3 ppm.

The term “ultrafiltration” as used herein refers to a pressure-drivenprocess wherein a mixture such as a solution, e.g. containing a proteinof interest, is passed through a membrane for concentration orpurification purposes. Ultrafiltration membranes typically have a meanpore size between 1 and 50 nm, which is between the mean pore size ofreverse osmosis and microfiltration membranes. The pore size is usuallyquantified by its ability to retain proteins of certain molecularweights and is normally quoted in terms of a nominal molecular weightcut off (NMWCO) in kDa. Ultrafiltration separates solutes based ondifferences in the rate of filtration of different substances across themembrane in response to a given pressure driving force which rate isdependent on the size of the solute. Thus, the solutes in the mixture orsolution are separated on the bases of size differences. Ultrafiltrationis frequently used in downstream processing for protein concentration,buffer exchange and desalting, protein purification, virus clearance,and clarification. The term “ultrafiltration” includes tangential flowfiltration (TFF) whereby the mixture such as solution is passedhorizontally along the ultrafiltration membrane. The term“ultrafiltration” does not include high performance tangential flowfiltration (HPTFF) whereby the solutes are separated not just on thebasis of size, but size and charge.

The term “total protein” as used herein, refers essentially all proteinsin a sample, including protein fragments of any size. Frequently, inconnection with periplasmic cell extract or other cell culture harvest,“total protein” refers to both HCPs and heterologous protein expressedin the cell culture contained in a sample. Total protein can bedetermined using methods that are well known in the art.

An antibody fragment that can be purified in accordance with the methodsof the present invention can be produced by culturing host cellstransformed with one or more expression vectors encoding the recombinantantibody fragment. The host cells are preferably prokaryotic cells,preferably gram-negative bacteria. More preferably, the host cells areE. coli cells. Prokaryotic host cells for protein expression are wellknown in the art (Terpe, 2006). The host cells are recombinant cellswhich have been genetically engineered to produce the protein ofinterest such as an antibody fragment. The recombinant E. coli hostcells may be derived from any suitable E. coli strain including fromMC4100, TG1, TG2, DHB4, DH5α, DH1, BL21, K12, XL1Blue and JM109. Oneexample is E. coli strain W3110 (ATCC 27,325) a commonly used hoststrain for recombinant protein fermentations. Antibody fragments canalso be produced by culturing modified E. coli strains, for examplemetabolic mutants or protease deficient E. coli strains.

An antibody fragment that can be purified in accordance with the methodsof the present invention is typically found in either the periplasm ofthe E. coli host cell or in the host cell culture supernatant, dependingon the nature of the protein, the scale of production and the E. colistrain used. The methods for targeting proteins to these compartmentsare well known in the art (Makrides, 1996). Examples of suitable signalsequences to direct proteins to the periplasm of E. coli include the E.coli PhoA, OmpA, OmpT, LamB and OmpF signal sequences. Proteins may betargeted to the supernatant by relying on the natural secretory pathwaysor by the induction of limited leakage of the outer membrane to causeprotein secretion examples of which are the use of the pelB leader, theprotein A leader, the co-expression of bacteriocin release protein, themitomycin-induced bacteriocin release protein along with the addition ofglycine to the culture medium and the co-expression of the kil gene formembrane permeabilization. Most preferably, in the methods of theinvention, the recombinant protein is expressed in the periplasm of thehost E. coli.

Expression of the recombinant protein in the E. coli host cells may alsobe under the control of an inducible system, whereby the expression ofthe recombinant antibody in E. coli is under the control of an induciblepromoter. Many inducible promoters suitable for use in E. coli are wellknown in the art and depending on the promoter; expression of therecombinant protein can be induced by varying factors such astemperature or the concentration of a particular substance in the growthmedium. Examples of inducible promoters include the E. coli lac, tac,and trc promoters which are inducible with lactose or thenon-hydrolyzable lactose analog, isopropyl-b-D-1-thiogalactopyranoside(IPTG) and the phoA, trp and araBAD promoters which are induced byphosphate, tryptophan and L-arabinose respectively. Expression may beinduced by, for example, the addition of an inducer or a change intemperature where induction is temperature dependent. Where induction ofrecombinant protein expression is achieved by the addition of an inducerto the culture the inducer may be added by any suitable method dependingon the fermentation system and the inducer, for example, by single ormultiple shot additions or by a gradual addition of inducer through afeed. It will be appreciated that there may be a delay between theaddition of the inducer and the actual induction of protein expressionfor example where the inducer is lactose there may be a delay beforeinduction of protein expression occurs while any pre-existing carbonsource is utilized before lactose.

E. coli host cell cultures (fermentations) may be cultured in any mediumthat will support the growth of E. coli and expression of therecombinant protein. The medium may be any chemically defined mediumsuch as e.g. described in (Durany O, 2004).

Culturing of the E. coli host cells can take place in any suitablecontainer such as a shake flask or a fermenter depending on the scale ofproduction required. Various large scale fermenters are available with acapacity of more than 1,000 liters up to about 100,000 liters.Preferably, fermenters of 1,000 to 50,000 liters are used, morepreferably 1,000 to 10,000 or 12,000 liters. Smaller scale fermentersmay also be used with a capacity of between 0.5 and 1,000 liters.

Fermentation of E. coli may be performed in any suitable system, forexample continuous, batch or fed-batch mode depending on the protein andthe yields required. Batch mode may be used with shot additions ofnutrients or inducers where required. Alternatively, a fed-batch culturemay be used and the cultures grown in batch mode pre-induction at themaximum specific growth rate that can be sustained using the nutrientsinitially present in the fermenter and one or more nutrient feed regimesused to control the growth rate until fermentation is complete.Fed-batch mode may also be used pre-induction to control the metabolismof the E. coli host cells and to allow higher cell densities to bereached.

If desired, the host cells may be subject to collection from thefermentation medium, e.g. host cells may be collected from the sample bycentrifugation, filtration or by concentration. In particular, themethods of the invention are suitable for the large-scale industrialmanufacture of antibodies of therapeutic quality.

In one embodiment the process according to the present inventioncomprises prior to the cation exchange chromatography capture step astep of centrifugation of cell culture harvest, followed by suspensionof the host cells by addition of the extraction buffer.

For E. coli fermentation processes wherein the protein of interest suchas an antibody fragment is found in the periplasmic space of the hostcell it is required to release the protein from the host cell. Therelease may be achieved by any suitable method such as cell lysis bymechanical or pressure treatment, freeze-thaw treatment, osmotic shock,extraction agents or heat treatment. Such extraction methods for proteinrelease are well known in the art.

In a preferred embodiment an extraction buffer is added to the sampleand the sample is then subjected to a heat treatment step. The heattreatment step is preferably as described in detail in U.S. Pat. No.5,655,866. The heat treatment step makes it possible to obtain a sampleof soluble, correctly folded and assembled antibody fragment byfacilitating the removal of other antibody-related material.

The heat treatment step is performed by subjecting the sample to adesired elevated temperature. Most preferably, heat treatment step isperformed within the range of 30° C. to 70° C. The temperature can beselected as desired and may depend on the stability of the antibody forpurification. In another embodiment, the temperature is within the range40° C. to 65° C., or preferably within the range 40° C. to 60° C., morepreferably within the range 45° C. to 60° C., even more preferablywithin the range 50° C. to 60° C. and most preferably at 55° C. to 60°C., 58° C. to 60° C. or 59° C. Thus, the minimum temperatures are 30°C., 35° C. or 40° C. and the maximum temperatures 60° C., 65° C. or 70°C.

The heat treatment step is preferably carried out for a prolonged periodof time. The length of heat treatment is preferably between 1 and 24hours, more preferably between 4 and 18 hours, even more preferablybetween 6 and 16 hours and most preferably between 10 and 14 hours orbetween 10 and 12 hours, for example 12 hours. Thus, the minimum timefor heat treatment is 1, 2 or 3 hours and the maximum is 20, 22 or 24hours.

In a particular embodiment, the heat treatment is performed at 50° C. to60° C. for 10 to 16 hours, and more preferably at 59° C. for 10 to 12hours. One skilled in the art will understand that temperatures and timecan be selected as suits the sample in question and the characteristicsof the antibody being produced.

Following the step of extraction the mixture containing the protein ofinterest such an antibody fragment may be subjected to a step ofcentrifugation and/or filtration prior to the step of adjusting the pH.

In further embodiments the process according to any of the embodimentsof the first or second aspect of the invention is performed with anantibody fragment, for example a Fab or s Fab′, that binds specificallyto VEGF-A, glycoprotein IIb/IIIa receptor, C5, HER2/neu, TNFα, IL1β,CD40-L, OX40 or ICOS.

In a preferred embodiment of the invention the process according to anyof the embodiments of the first or second aspect of the invention isperformed with periplasmic cell extract comprising an antibody fragmentwhich is an antibody fragment having specificity for human TNFα, morepreferably CDP870, as described in WO 01/094585 (the contents of whichare incorporated herein by reference).

In a one embodiment the antibody fragment having specificity for humanTNFα, comprises a heavy chain wherein the variable domain comprises aCDR having the sequence shown in SEQ ID NO:1 for CDRH1, the sequenceshown in SEQ ID NO:2 for CDRH2 or the sequence shown in SEQ ID NO:3 forCDRH3.

In one embodiment the antibody fragment comprises CDRs having thesequence shown in SEQ ID NO:4 for CDRL1, the sequence shown in SEQ IDNO:5 for CDRL2 or the sequence shown in SEQ ID NO:6 for CDRL3.

In one embodiment the antibody fragment comprises CDRs having thesequence shown in SEQ ID NO:1 for CDRH1, the sequence shown in SEQ IDNO:2 for CDRH2 or the sequence shown in SEQ ID NO:3 for CDRH3 and CDRshaving the sequence shown in SEQ ID NO:4 for CDRL1, the sequence shownin SEQ ID NO:5 for CDRL2 or the sequence shown in SEQ ID NO:6 for CDRL3.

In one embodiment the antibody fragment comprises SEQ ID NO:1 for CDRH1,SEQ ID NO: 2 for CDRH2, SEQ ID NO:3 for CDRH3, SEQ ID NO:4 for CDRL1,SEQ ID NO:5 for CDRL2 and SEQ ID NO:6 for CDRL3.

The antibody fragment is preferably a CDR-grafted antibody fragmentmolecule and typically the variable domain comprises human acceptorframework regions and non-human donor CDRs.

Preferably, the antibody fragment comprises the light chain variabledomain CDP870 (SEQ ID NO:7) and the heavy chain variable domain CDP870(SEQ ID NO:8).

It is preferred that the antibody fragment is a modified Fab fragmentwherein the modification is the addition to the C-terminal end of itsheavy chain one or more amino acids to allow the attachment of aneffector or reporter molecule. Preferably, the additional amino acidsform a modified hinge region containing one or two cysteine residue towhich the effector or reporter molecule may be attached. Such a modifiedFab fragment preferably has a heavy chain comprising or consisting ofthe sequence given as SEQ ID NO:10 and the light chain comprising orconsisting of the sequence given as SEQ ID NO:9.

In further embodiments the process according to any of the embodimentsof the first or second aspect of the invention is performed withabciximab, ranibizumab, pexelizumab, CDP484, or CDP7657.

Equilibration

In further embodiments of the invention the cation exchangechromatography column for the primary capture step is equilibrated withan anionic buffer of suitable composition to buffer at the required pHand conductivity (for example 50 mM sodium acetate at pH 5 or 50 mMsodium lactate at pH 4.0. The equilibration can be achieved using atleast 2 column volumes of the equilibration buffer, but may also includea two step equilibration process with two column volumes of a buffercontaining 1 M NaCl (to ensure the column is adequately charged with therelevant counter cation) followed by at least 2 column volumes of theequilibration buffer.

In further embodiments of the invention the anion exchangechromatography column of the second chromatography step is equilibratedin the same way except that the equilibration buffer is ideally acationic buffer e.g. 20 mM Tris HCl at pH 8.0 or 20 mM bis-Tris HCl atpH 7.0. The equilibration of the anion exchange chromatography columncan also be achieved using a two step equilibration process with buffercontaining 1 M NaCl (to ensure the column is adequately charged with therelevant counter anion) followed by the equilibration buffer; or asingle step directly with the equilibration buffer.

Washing

In further embodiments of the invention after loading the cationexchange chromatography column for the primary step, it is washed withat least 2 column volumes of the equilibration buffer. Additionalimpurity may be removed by using a wash buffer with a higherconductivity, e.g. 0.5 mScm or higher conductivity.

In further embodiments of the invention after loading the anion exchangechromatography column for the second chromatography step, the column iswashed with up to 2 column volumes of the equilibration buffer. Otherbuffers with similar pH and conductivity may also be used. Buffers witha higher conductivity are not recommended if the maximum removal of theprocess-related impurities is desired.

Elution

In further embodiments of the invention the antibody fragment is elutedfrom the cation exchange chromatography column in the primary capturestep using a buffer either at a higher pH, a higher conductivity or acombination of the two. The increased conductivity can be achieved withthe addition of NaCl (or other salt) to the equilibration buffer at aconcentration of greater than 50 mM, preferably greater than 100 mM andeven more preferably greater than 200 mM.

In further embodiments of the invention the elution of the antibodyfragment from the anion exchange chromatography column in the secondchromatography step occurs during the loading and wash steps. The boundprocess-related impurities can be eluted by using a buffer either at alower pH, higher conductivity or a combination of both. Ideally, thehigher conductivity should be at least 70 mS/cm.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation. While this invention has been described in connectionwith specific embodiments thereof, it will be understood that it iscapable of further modifications. This application is intended to coverany variations, uses or adaptations of the invention following, ingeneral, the principles of the invention and including such departuresfrom the present disclosure as come within known or customary practicewithin the art to which the invention pertains and as may be applied tothe essential features hereinbefore set forth follows in the scope ofthe appended claims.

As used herein, “a” or “an” may mean one or more. The use of the term“or” herein is used to mean “and/or” unless explicitly indicated torefer to alternatives only or the alternatives are mutually exclusive,although the disclosure supports a definition that refers to onlyalternatives and “and/or.” As used herein “another” may mean at least asecond or more.

As used herein, “between X and Y” may mean a range including X and Y.

All references cited herein, including journal articles or abstracts,published or unpublished U.S. or foreign patent application, issued U.S.or foreign patents or any other references, are entirely incorporated byreference herein, including all data, tables, figures and text presentedin the cited references. Additionally, the entire contents of thereferences cited within the references cited herein are also entirelyincorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

EXAMPLES Example 1

CDP870 Fab′ was expressed as a heterologous protein in E. coli W3110host cells at a concentration of 1.9 g/L. The heterologous protein wasreleased from the periplasmic space of the host cells by the addition ofTris-EDTA and heat treatment at 50° C. Cellular material was removedthrough centrifugation and the cell extract containing the heterologousprotein was adjusted through addition of acetic acid to a pH 4.5. The pHadjusted cell extract was then clarified using a combination ofcentrifugation and depth filtration with 0.2 μm filtration.

The clarified extract (feed stream) was diluted with a dilution factorof 4 with water to achieve a conductivity of 3.5 mS/cm.

The feed stream containing the CDP870 Fab′ was then loaded to about 75g/L resin at a flow rate of 400 cm/h onto a Capto S™ cation exchangecolumn from GE Healthcare [highly cross-linked rigid agarose bead (meanparticle size 90 μm) with a sulphonate cation exchange ligand attachedvia a dextran linker (ionic capacity 0.11-0.14 mmol Na⁺/ml)]. The CaptoS™ cation exchange column had a column bed height of 20 cm with adiameter of 7.7 mm. The column had been equilibrated prior to feedstream loading with 6 column volumes of 50 mM sodium acetate bufferadjusted to pH 4.5 with acetic acid.

After loading the column was washed with 5 column volumes of the 50 mMsodium acetate buffer adjusted to pH 4.5 with acetic acid, until the allthe unbound material is washed off. The CDP870 Fab′ fraction was elutedwith a 20 column volumes gradient up to a concentration of 50 mM sodiumacetate and 250 mM NaCl adjusted to pH 4.5 with acetic acid. The elutedCDP870 Fab′ was collected when the UV absorbance at 280 nm exceeded 0.26AU/cm until it dropped below 0.54 AU/cm.

The CDP870 Fab′ pool was subjected to ultrafiltration in a centrifugalconcentrator using a polyether sulphone-based ultrafiltration membranewith a nominal molecular weight cut-off of 10 kDa resulting in aconcentration of the CDP870 Fab′ pool to 6 mg/mL. Subsequentlydiafiltration was performed until the CDP870 Fab′ pool in 20 mM Tris hadreached pH 8.0 and a conductivity of 1.1 mS/cm. 6 volumes of buffer wererequired for the diafiltration.

The CDP870 Fab′ pool was loaded onto a Capto™ anion exchangechromatography column from GE Healthcare [highly cross-linked rigidagarose bead (mean particle size 90 μm) with a quaternary amine anionexchange ligand attached via a dextran linker (ionic capacity 0.16-0.22mmol Cl⁻/ml)] at a flow rate of 500 cm/h and applying about 5 g ofCDP870 Fab′ per liter of resin. The Capto Q™ anion exchange column had acolumn bed height of 10 cm with a diameter of 7.7 mm. The column hadbeen equilibrated prior to the anion exchange chromatography step with 3column volumes of 20 mM Tris/1M NaCl buffer adjusted to pH 8.0 withhydrochloric acid and 5 column volumes 20 mM Tris buffer adjusted to pH8.0 with hydrochloric acid.

The column was then washed with 5 column volumes of equilibration bufferto recover the CDP870 Fab′.

Collection of the CDP870 Fab′ fraction started when the UV absorbance at280 nm exceeded 0.5 AU/cm shortly after the start of the loading. Theend of the fraction collection occurred during the wash when the UVabsorbance dropped below 0.5 AU/cm. The yield for the antibody fragmenton this step was 90.5%.

Example 2

CDP870 Fab′ was expressed as a heterologous protein in E. coli hostcells at a concentration of 1.9 g/L. The heterologous protein wasreleased from the periplasmic space of the host cells by the addition ofTris-EDTA and heat treatment at 50° C. Cellular material was removedthrough centrifugation and the cell extract containing the heterologousprotein was adjusted through addition of acetic acid to a pH 4.5. The pHadjusted cell extract was then clarified using a combination ofcentrifugation and depth filtration with 0.2 μm filtration.

The clarified extract (feed stream) was diluted with a dilution factorof 4 with water to achieve a conductivity of 3.5 mS/cm.

The feed stream containing the CDP870 Fab′ was then loaded to about 51g/L resin at a flow rate of 300 cm/h onto a Capto S cation exchangecolumn from GE Healthcare [highly cross-linked rigid agarose bead (meanparticle size 90 μm) with a sulphonate cation exchange ligand attachedvia a dextran linker (ionic capacity 0.11-0.14 mmol Na⁺/ml)]. The CaptoS cation exchange column had a column bed height of 20 cm with adiameter of 16 mm. The column had been equilibrated prior to feed streamloading with 3 column volumes of 50 mM sodium acetate/1 M NaCl bufferadjusted to pH 4.5 with acetic acid and subsequently 4 column volumes of50 mM sodium acetate buffer adjusted to pH 4.5 with acetic acid.

After loading the column was washed with 6 column volumes of the 50 mMsodium acetate buffer adjusted to pH 4.5 with acetic acid, until the allthe unbound material is washed off. The CDP870 Fab′ fraction was elutedwith up to 8 column volumes of buffer with 50 mM sodium acetate and 200mM NaCl adjusted to pH 4.5 with acetic acid. The eluted CDP870 Fab′ wascollected when the UV absorbance at 280 nm exceeded 0.5 AU/cm until itdropped below 0.5 AU/cm.

The CDP870 Fab′ pool was subjected to ultrafiltration in a centrifugalconcentrator (Amicon) using a polyether sulphone-based ultrafiltrationmembrane with a nominal molecular weight cut-off of 10 kD resulting in aconcentration of the CDP870 Fab′ pool to 18 mg/mL. Subsequentlydiafiltration was performed until the CDP870 Fab′ pool in 20 mM Tris hadreached pH 8.3 and a conductivity of 1.0 mS/cm. 6 volumes of buffer wererequired for the diafiltration.

The CDP870 Fab′ pool was loaded onto a Capto Q anion exchangechromatography column from GE Healthcare [highly cross-linked rigidagarose bead (mean particle size 90 μm) with a quaternary amine anionexchange ligand attached via a dextran linker (ionic capacity 0.16-0.22mmol Cl⁻/ml)] at a flow rate of 250 cm/h and applying about 30 g ofCDP870 Fab′ per liter of resin. The Capto™ anion exchange column had acolumn bed height of 10 cm with a diameter of 7.7 mm. The column hadbeen equilibrated prior to the anion exchange chromatography step with 3column volumes of 20 mM Tris/1M NaCl buffer adjusted to pH 8.3 withhydrochloric acid and 5 column volumes 20 mM Tris buffer adjusted to pH8.3 with hydrochloric acid.

The column was then washed with 5 column volumes of equilibration bufferto recover the CDP870 Fab′.

Collection of the CDP870 Fab′ fraction started when the UV absorbance at280 nm exceeded 0.5 AU/cm shortly after the start of the loading. Theend of the fraction collection occurred during the wash when the UVabsorbance dropped below 2.0 AU/cm.

Example 3

CDP870 Fab′ was expressed as a heterologous protein in E. coli hostcells at a concentration of 2.5 g/L. The heterologous protein wasreleased from the periplasmic space of the host cells by the addition ofTris-EDTA and heat treatment at 50° C. Cellular material was removedthrough centrifugation and the cell extract containing the heterologousprotein was adjusted through addition of acetic acid to a pH 4.5. The pHadjusted cell extract was then clarified using a combination ofcentrifugation and depth filtration with 0.2 μm filtration.

The clarified extract (feed stream) was diluted with a dilution factorof 4 with water to achieve a conductivity of 4.0 mS/cm.

The feed stream containing the CDP870 Fab′ was then loaded to about 60g/L resin at a flow rate of 400 cm/h onto a Capto S cation exchangecolumn from GE Healthcare [highly cross-linked rigid agarose bead (meanparticle size 90 μm) with a sulphonate cation exchange ligand attachedvia a dextran linker (ionic capacity 0.11-0.14 mmol Na⁺/ml)]. The CaptoS cation exchange column had a column bed height of 24 cm with adiameter of 20 cm. The column had been equilibrated prior to feed streamloading with 3 column volumes of 50 mM sodium acetate/1 M NaCl bufferadjusted to pH 4.5 with acetic acid and subsequently 4 column volumes of50 mM sodium acetate buffer adjusted to pH 4.5 with acetic acid.

After loading the column was washed with 6 column volumes of the 50 mMsodium acetate buffer adjusted to pH 4.5 with acetic acid, until the allthe unbound material is washed off. The CDP870 Fab′ fraction was elutedwith up to 5 column volumes of buffer with 50 mM sodium acetate and 250mM NaCl adjusted to pH 4.5 with acetic acid. The eluted CDP870 Fab′ wascollected when the UV absorbance at 280 nm exceeded 1.75 AU/cm until itdropped below 0.55 AU/cm.

The CDP870 Fab′ was subjected to ultrafiltration in tangential flow modeusing 0.4 m² polyethersulphone membrane with a 10 kDa nominal molecularweight cut-off (Pall 10k Omega Centramate T-Series). The CDP870 Fab′ wasconcentrated to 51 mg/mL before diafiltration with 5.8 volumes of 20 mMTris pH 8.5 until the pH of the Fab′ solution was pH 8.5 and theconductivity was 0.8 mS/cm. The Fab′ was recovered from theultrafiltration equipment and pooled with 600 mL of 20 mM Tris pH 8.5buffer used to wash any remaining Fab′ from the system.

The CDP870 Fab′ pool was loaded onto a Capto Q anion exchangechromatography column from GE Healthcare [highly cross-linked rigidagarose bead (mean particle size 90 μm) with a quaternary amine anionexchange ligand attached via a dextran linker (ionic capacity 0.16-0.22mmol Cl⁻/ml)] at a flow rate of 200 cm/h and applying about 37 g ofCDP870 Fab′ per liter of resin. The Capto Q™ anion exchange column had acolumn bed height of 24 cm with a diameter of 20 cm. The column had beenequilibrated prior to the anion exchange chromatography step with 3column volumes of 20 mM Tris/1M NaCl buffer adjusted to pH 8.5 withhydrochloric acid and 5 column volumes 20 mM Tris buffer adjusted to pH8.5 with hydrochloric acid.

The column was then washed with 5 column volumes of equilibration bufferto recover the CDP870 Fab′.

Collection of the CDP870 Fab′ fraction started when the UV absorbance at280 nm exceeded 0.5 AU/cm shortly after the start of the loading. Theend of the fraction collection occurred during the wash when the UVabsorbance dropped below 2.0 AU/cm.

Example 4 REFERENCE LIST

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The invention claimed is:
 1. A process for the purification of anantibody fragment from periplasmic cell extract comprising: a) a firstchromatography step to capture the antibody fragment by subjecting amixture comprising the antibody fragment and bacterial host cellprotein, wherein bacterial host cell protein is present at 200μg/ml-10,000 μg/ml and wherein the antibody fragment is at aconcentration of at least 1.5 g/L in said mixture, to cation exchangechromatography and subsequent elution, thereby producing a first eluatecontaining the antibody fragment; and b) a second chromatography stepwherein the first eluate is subjected to anion exchange chromatographyto capture impurities and produce a flow through containing the antibodyfragment.
 2. The process according to claim 1, wherein the processcomprises not more than two chromatography steps.
 3. The processaccording to claim 1, wherein all chromatography steps are performed ona chromatography column.
 4. The process according to claim 1, whereinthe cation exchange chromatography is performed in elution mode.
 5. Theprocess according to claim 1, wherein the first cation chromatographystep comprises the following steps in sequential order: a) loading amixture containing an antibody fragment onto the cation exchange column,b) washing the cation exchange column with a wash buffer wherein duringthe washing the conductivity, pH and salt concentration of the bufferremains essentially unchanged, and c) eluting the antibody fragment withan elution buffer.
 6. The process according to claim 5, wherein the pHof the washing buffer is identical to the pH of the mixture containingan antibody fragment prior to first chromatography step.
 7. The processaccording to claim 5, wherein the mixture containing an antibodyfragment, prior to the first chromatography step has a pH of between 4.0to 5.0.
 8. The process according to claim 5, wherein the antibodyfragment is eluted with a buffer that has a higher pH, a higherconductivity or both a higher pH and higher conductivity than the washbuffer.
 9. The process according to claim 1, wherein the cation exchangechromatography in the first chromatography step is performed at a flowrate of at least 300 cm/h.
 10. The process according to claim 1, whereinthe cation exchange chromatography in the first chromatography step isperformed at a conductivity of not more 6 mS/cm.
 11. The processaccording to claim 1, wherein the cation exchange chromatography in thefirst chromatography step is performed in a chromatography columncomprising sulphonyl, sulphopropyl or carboxymethyl coupled to a resin.12. The process according to claim 1, wherein the cation exchangechromatography column in the first chromatography step has a dynamicbinding capacity for the antibody fragment of between 50 and 75 g/Lresin.
 13. The process according to claim 11, wherein the cationexchange chromatography column resin in the first chromatography stephas a mean particular size of at least 50 μm.
 14. The process accordingto claim 1, wherein in the cation exchange chromatography of the primarycapture step between 5 and 100 g antibody fragment per liter resin areloaded.
 15. The process according to claim 1, wherein the anion exchangechromatography in the second chromatography step is performed on a resincomprising quaternary ammonium (Q), diethylaminoethyl (DEAF) ortrimethylaminoethyl (TMAE).
 16. The process according to claim 1,wherein the antibody fragment is a Fab, Fab′ or scFv.
 17. The processaccording to claim 16, wherein the Fab or Fab′ binds specifically toVEGF-A, FeRn, OX40, glycoprotein IIb/IIIa receptor, C5, HER2/neu, TNFα,IL1β or CD40-L.
 18. The process according to claim 17, wherein the Fabor Fab′ is abciximab, ranibizumab, pexelizumab, CDP870 or CDP7657. 19.The process according to claim 1, wherein the antibody fragmentrecovered from the process contains host cell protein in an amount ofnot more than 150 parts per million.
 20. The process according to claim1, said process further comprising ultrafiltration of the first eluateand ultrafiltration of the flow through containing the antibodyfragment.